Intraperitoneal Injection Of Phosphate-buffered Saline Research Articles

PAR1 Is a Candidate Target for the Treatment of Peritoneal Dissemination in Gastric Cancer.

Peritoneal dissemination (PD) is a frequent cause of death in gastric cancer (GC), and there is evidence of an association between protease-activated receptor-1 (PAR1) and the development of PD. This study hypothesized that PD in GC might be influenced by PAR1. The cytotoxic effect of paclitaxel (PTX) on PAR1-transfected MKN45 (MKN45/PAR1) cells was analyzed using the MTT assay, and IC50 values were determined. In female athymic nude mice, MKN45/PAR1 cells were suspended in 0.05 ml phosphate-buffered saline (PBS) medium and inoculated into the stomach mid-wall. In each group, intraperitoneal injections of PBS, PTX, SCH79797 (PAR1-antagonist), or PTX plus SCH79797 were administered on days 8, 15, and 22 following tumor inoculation. At 56 days after tumor inoculation, mice were examined for both abdominal tumor nodule status and size and weight of the tumors. The IC50 of PTX for MKN45/PAR1 cells was 0.0697 μM and that of SCH79797 was 0.0145 μM. Mean survival of the MKN45/PAR1 mice in the PBS group was 28.75 days, whereas survival times for the mice treated with SCH79797, PTX, or a combination of PTX and SCH79797 were 31.2, 49.2, and 48.5 days, respectively. Tumor weight was smaller in the group receiving PTX and SCH79797 intraperitoneally compared with that in the PBS group (1,086±127.2 mg vs. 33.2±19.9 mg; p<0.001). The PAR1 antagonist was found to inhibit PD in a PAR1-expressing GC cell line. PAR1 may serve as a promising therapeutic target for managing PD in gastric cancer, as it plays a crucial role in its progression.

Halofuginone prevents outer retinal degeneration in a mouse model of light-induced retinopathy.

Photoreceptor cell death can cause progressive and irreversible visual impairments. Still, effective therapies on retinal neuroprotection are not available. Hypoxia-inducible factors (HIFs) are transcriptional factors which strongly regulate angiogenesis, erythropoiesis, intracellular metabolism, and programed cell death under a hypoxic or an abnormal metabolic oxidative stress condition. Therefore, we aimed to unravel that inhibition of HIFs could prevent disease progression in photoreceptor cell death, as recent studies showed that HIFs might be pathologic factors in retinal diseases. Adult male balb/cAJcl (8 weeks old; BALB/c) were used to investigate preventive effects of a novel HIF inhibitor halofuginone (HF) on a murine model of light-induced retinopathy. After intraperitoneal injections of phosphate-buffered saline (PBS) or HF (0.4 mg/kg in PBS) for 5 days, male BALB/c mice were subjected to a dark-adaption to being exposed to a white LED light source at an intensity of 3,000 lux for 1 hour in order to induce light-induced retinal damage. After extensive light exposure, retinal damage was evaluated using electroretinography (ERG), optical coherence tomography (OCT), and TUNEL assay. Light-induced retinal dysfunction was suppressed by HF administration. The amplitudes of scotopic a-wave and b-wave as well as that of photopic b-wave were preserved in the HF-administered retina. Outer retinal thinning after extensive light exposure was suppressed by HF administration. Based on the TUNEL assay, cell death in the outer retina was seen after light exposure. However, its cell death was not detected in the HF-administered retina. Halofuginone was found to exert preventive effects on light-induced outer retinal cell death.

Regulation of sexually dimorphic placental adaptation in LPS exposure-induced intrauterine growth restriction

BackgroundSexual dimorphism in placental physiology affects the functionality of placental adaptation during adverse pregnancy. Defects of placental function compromise fetal programming, affecting the offspring’s adult life. However, studies focusing on the relationship between sex-specific placental adaptation and consequent fetal maldevelopment under sub-optimal uterus milieu are still elusive.MethodsHere, we investigated the effects of maternal lipopolysaccharide (LPS) exposure between placental sex. Pregnant ICR mice received intraperitoneal injection of phosphate-buffered saline or 100, 200, and 400 µg/kg LPS on the gestational day (GD) 15.5. To determine whether prenatal maternal LPS exposure resulted in complicated pregnancy outcomes, survival rate of embryos was calculated and the growth of embryos and placentas was examined. To elucidate global transcriptomic changes occurring in the placenta, total RNA-sequencing (RNA-seq) was performed in female and male placentas.ResultsLPS administration induced placental inflammation in both sexes at GD 17.5. Prenatal infection resulted in growth retardation in both sexes of embryos, and especially more prevalently in male. Impaired placental development was observed in a sex-specific manner. LPS 400 µg/kg reduced the percentage area of the labyrinth in females and junctional zone in males, respectively. RNA-sequencing revealed widespread sexually dimorphic transcriptional changes in placenta. In particular, representative changes were involved in biological processes such as trophoblast differentiation, nutrient/ion transporter, pregnancy, and immune system.ConclusionsOur results present the sexually dimorphic responses of placental physiology in intrauterine growth restriction model and provide tentative relationship further to be elucidated between sex-biased placental functional change and long-term effects on the offspring’s later life.

Evaluation of experimental animal behaviors through establishment of an ovalbumin-induced experimental mouse model of allergic rhinitis

Allergic rhinitis (AR) was also called hay fever which was a type of nasal inflammation when the immune system overreacts to environmental allergen exposures. AR’s clinical symptoms included a runny or stuffy nose, sneezing, red, itchy, watery eyes, and eye swelling. The fluid in the nasal cavity was usually clear. Patients with AR can affect sleep and work qualities. Seriously, the AR symptoms can also cause asthma, allergic conjunctivitis, or atopic dermatitis. Therefore, it is an important issue to attenuate AR symptoms and research the novel therapeutic drugs for AR patients. The purpose of this study was to introduce an easy-to-establish experimental mouse model of AR. In this study, the male BALB/c mice were divided respectively into as the Group A (n = 12) and the Group B (n = 12). Group A and Group B were designed as the normal control and RA, respectively. BALB/c mice in Group B were sensitized by intraperitoneal injection of ovalbumin (OVA) on day 0, day 4, day 13, and day 20, followed by continuous nasal administration of OVA solution once per day between day 21-43. BALB/c mice in Group A received sensitization of intraperitoneal injection of phosphate-buffered saline (PBS) on day 0, day 4, day 13, and day 20 and continuous nasal administration of PBS instead of OVA once per day between day 21-43. Before and after sensitization, the frequencies of nasal symptoms (sneezing, nasal rubbing) were recorded and counted. Results were showed that sneezing times in Group B were higher than Group A on D29, D30, D36, and D43 of the experiment. The sneezing times in Group A were significant higher on D29 and D30 of the experiment. However, the sneezing times in Group B were significant higher on D29, D30, D36, and D43 of the experiment. The rubbing times in Group B were higher than Group A on D29, D30, D36, and D43 of the experiment. The rubbing times in Group A were significant higher on D30 and D43 of the experiment. However, the rubbing times in Group B were significant higher on D29, D30, D36, and D43 of the experiment. Based on these results, a successful mouse model of AR has been established. We hope that this RA mouse model will provide a tool for the research of the novel AR therapeutic drugs and apply these novel AR therapeutic drugs to attenuate the AR symptoms in AR patients in the future.

Amniotic fluid stem cell administration can prevent epithelial injury from necrotizing enterocolitis.

Stem cell therapy has been proven to rescue intestinal injury and stimulate intestinal regeneration in necrotizing enterocolitis (NEC). Specifically, stem cells derived from amniotic fluid (AFSCs) and mesenchymal stem cells (MSCs) derived from bone marrow have shown promising results in the treatment of experimental NEC. This study aims to examine the effects of AFSCs and MSCs on the prevention of intestinal injury during experimental NEC. Supernatants from AFSC and MSC cultures were collected to perform proteomic analysis. Prior to NEC induction, mice received intraperitoneal injections of phosphate-buffered saline (PBS), 2 × 106 AFSCs, or 2 × 106 MSCs. We found that AFSCs grew faster than MSCs. Proteomic analysis indicated that AFSCs are primarily involved in cell development and growth, while MSCs are involved in immune regulation. Administering AFSCs before NEC induction decreased NEC severity and mucosal inflammation. Intestinal proliferation and endogenous stem cell activation were increased after AFSC administration. However, administering MSCs before NEC induction had no beneficial effects. This study demonstrated that AFSCs and MSCs have different protein release profiles. AFSCs can potentially be used as a preventative strategy for neonates at risk of NEC, while MSCs cannot be used. AFSCs and MSCs have distinct protein secretory profiles, and AFSCs are primarily involved in cell development and growth, while MSCs are involved in immune regulation. AFSCs are unique in transiently enhancing healthy intestinal epithelial cell growth, which offers protection against the development of experimental NEC. The prevention of NEC via the administration of AFSCs should be evaluated in infants at great risk of developing NEC or in infants with early signs of NEC.

Effect and Mechanism of Treating Experimental Autoimmune Encephalomyelitis in Mice with Butylphthalide Combined with Bone Marrow Mesenchymal Stem Cells

To explore the efficacy and mechanism of using 3-n-butylphthalide (NBP) in combination with bone marrow mesenchymal stem cells (BMSCs) in the treatment of experimental autoimmune encephalomyelitis (EAE) in mice. Myelin oligodendrocyte glycoprotein (MOG35-55) was used for the induction and establishment of the EAE model in C57BL/6 mice. The mice were randomly assigned to the EAE group, which received intraperitoneal injection of phosphate-buffered saline (PBS), the NBP-treated EAE group, or the NBP group, which received intraperitoneal injection of NBP, the BMSCs transplantion EAE group, or the BMSCs group, which received BMSCs injected into the lateral ventricle and intraperitoneal injection of PBS, and the BMSCs and NBP combination treatment EAE group, or the BMSCs+NBP group, which received BMSCs injected into the lateral ventricle and intraperitoneal injection of NBP. Each group had 10 mice, while ten normal mice were used as the blank control group receiving intraperitoneal injection of PBS. The neurological function scores were documented daily. The mice were sacrificed 22 days after EAE induction, and the demyelination state of of the spinal cords was observed through Luxol fast blue (LFB) staining. In addition, the levels of serum interleukin-6 (IL-6), IL-10, IL-17, IL-22 and transforming growth factor-β (TGF-β) were examined with ELISA. The levels of glial fibrillary acidic protein (GFAP), microtubule associated protein-2 (MAP-2) and myelin basic protein (MBP) in the brain were examined with immunofluorescence staining. Western blot was used to check the expressions of nuclear factor (NF)-κB pathway, phosphoinositide-3 kinase (PI3K)/protein kinase B (PKB or Akt) pathway, IL-17 and forkhead box P3 (Foxp3) in the spinal cords. The neurological function scores and average scores of each treatment group were significantly lower than those of the EAE group ( P<0.05). The scores of the BMSCs+NBP group decreased more significantly than those of the single treatment groups (the NBP group and the BMSCs group) ( P<0.05). LFB staining results of the spinal cords were consistent with the neurological function scores and the average scores. Compared with the EAE group, the levels of pro-inflammatory cytokines, including IL-6, IL-17 and IL-22, significantly decreased ( P<0.05), and the levels of anti-inflammatory cytokines IL-10 and TGF-β significantly increased ( P<0.05). The change in cytokine expression was more significant in the BMSCs+NBP group ( P<0.05). The expressions of GFAP, MAP-2 and MBP in the BMSCs+NBP group were significantly higher than those of the BMSCs group ( P<0.05). Compared with the EAE group, the p-NF-κB/NF-κB ratio and the IL-17/Foxp3 ratio in NBP group, BMSCs group and BMSCs+NBP group decreased, while P-IκBα/IκBα, p-pI3k/PI3K and P-Akt/Akt ratios increased, especially in the BMSCs+NBP group( P<0.05). The combined treatment of NBP and BMSCs can help alleviate the symptoms of EAE model mice, showing better efficacy than treatment with NBP or BMSCs alone. The mechanism is related to the inhibition of the NF-κB pathway to regulate Th17/Foxp3 ratio and the activation of the PI3K/Akt pathway to promote the neurogenic differentiation of BMSCs.

Oridonin induces autophagy-mediated cell death in pancreatic cancer by activating the c-Jun N-terminal kinase pathway and inhibiting phosphoinositide 3-kinase signaling.

BackgroundOridonin is a diterpenoid isolated from Rabdosia rubescens that has potent anticancer activity. This study set out to investigate the antitumor effects of oridonin in pancreatic carcinoma (PC) and their underlying mechanisms.MethodsTo investigate the antitumor effects of oridonin, we developed an orthotopic C57BL/6 mouse model of PC. After successful establishment of the model, the mice were given a daily intraperitoneal injection of phosphate-buffered saline containing 0.1% dimethyl sulfoxide or oridonin for 2 weeks. In vitro experiments including MTT assay and flow cytometry were performed to examine cell viability and apoptosis. Panc-1 and Panc02 cells were transfected with a green fluorescent protein (GFP)-LC3 plasmid. After the cells had been treated with or without 20 μM oridonin and 10 μM 3-MA, the formation of GFP-LC3 puncta was detected by fluorescence microscopy. The levels of the autophagy-related proteins Beclin-1, LC3, and p62 were measured by western blotting.ResultsOridonin inhibited the proliferation of PC cells and induced their apoptosis in vitro and in vivo. Treatment with oridonin also led to an increase in the quantity of LC3B II protein and upregulation of the p62 and Beclin-1 levels in PC cells. The effects of oridonin on PC cell proliferation, apoptosis, and autophagy were mediated via the simultaneous inhibition of the phosphoinositide 3-kinase pathway and activation of the c-Jun N-terminal kinase pathway.ConclusionsOur study is the first to confirm the antitumor and autophagy-activating effects of oridonin on PC cells. In light of these results, oridonin may be a promising therapeutic agent for PC.

Administration of extracellular vesicles derived from human amniotic fluid stem cells: a new treatment for necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease. Amniotic fluid stem cells (AFSC) improve NEC injury but human translation remains difficult. We aimed to evaluate the use of extracellular vesicles (EV) derived from human AFSC. Human AFSC (hAFSC) were cultured according to the protocol (Celprogen Inc., California, U.S.A.). Conditioned medium was obtained, ultra-centrifuged, and EV were suspended in phosphate-buffered saline (PBS). C57BL/6 pups were grouped into: (1) breast-fed (Control, n = 11); (2) NEC + placebo (NEC + PBS; n = 10); and (3) NEC + treatment (NEC + EV; n = 11). NEC was induced post-natal days P5-9 by (A) gavage feeding hyperosmolar formula; (B) hypoxia for 10min; and (C) lipopolysaccharide. Intra-peritoneal injections of PBS or hAFSC-EV were given on P6-7. All animals were sacrificed on P9 and terminal ileum harvested. hAFSC-EV administration reduced intestinal injury (p = 0.0048), NEC incidence (score ≥ 2), and intestinal inflammation (IL-6 p < 0.0001; TNF-α p < 0.0001). Intestinal stem cell expression (Lgr5 +) and cellular proliferation (Ki67) were enhanced above control levels following hAFSC-EV administration (Lgr5 p = 0.0003; Ki67 p < 0.0001). hAFSC-EV administration reduced intestinal NEC injury and inflammation while increasing stem cell expression and cellular proliferation. hAFSC-EV administration may induce similar beneficial effects to exogenous stem cells.

Effect of post-exercise lactate administration on glycogen repletion and signaling activation in different types of mouse skeletal muscle.

Lactate is not merely a metabolic intermediate that serves as an oxidizable and glyconeogenic substrate, but it is also a potential signaling molecule. The objectives of this study were to investigate whether lactate administration enhances post-exercise glycogen repletion in association with cellular signaling activation in different types of skeletal muscle. Eight-week-old male ICR mice performed treadmill running (20 m/min for 60 min) following overnight fasting (16 h). Immediately after the exercise, animals received an intraperitoneal injection of phosphate-buffered saline or sodium lactate (equivalent to 1 g/kg body weight), followed by oral ingestion of water or glucose (2 g/kg body weight). At 60 min of recovery, glucose ingestion enhanced glycogen content in the soleus, plantaris, and gastrocnemius muscles. In addition, lactate injection additively increased glycogen content in the plantaris and gastrocnemius muscles, but not in the soleus muscle. Nevertheless, lactate administration did not significantly alter protein levels related to glucose uptake and oxidation in the plantaris muscle, but enhanced phosphorylation of TBC1D1, a distal protein regulating GLUT4 translocation, was observed in the soleus muscle. Muscle FBP2 protein content was significantly higher in the plantaris and gastrocnemius muscles than in the soleus muscle, whereas MCT1 protein content was significantly higher in the soleus muscle than in the plantaris and gastrocnemius muscles. The current findings suggest that an elevated blood lactate concentration and post-exercise glucose ingestion additively enhance glycogen recovery in glycolytic phenotype muscles. This appears to be associated with glyconeogenic protein content, but not with enhanced glucose uptake, attenuated glucose oxidation, or lactate transport protein.

Therapeutic potential of xanthones from Swertia chirata in breast cancer cells.

Background & objectives:Medicinal plants like Swertia chirata are rich sources of different xanthones. This study was aimed to assess the cytotoxic potential of four most abundant xanthones present in S. chirata both in vivo and in vitro in Ehrlich ascites carcinoma (EAC), a mouse transplantable breast carcinoma cell line and two human breast carcinoma cell lines (MCF-7 and MDA-MB-231).Methods:Four xanthones derived from S. chirata namely 1-hydroxy-3,7,8-trimethoxyxanthone (XA), 1,8-dihydroxy-3,5-dimethoxyxanthone (XB), 1-hydroxy-3,5,8-trimethoxyxanthone (XC) and 1,5,8-trihydroxy-3-methoxyxanthone (XD) were used for determination of sub-lethal dose on the cell lines EAC, MCF-7, MDA-MB-231 and verified toxicity of sub-lethal dose on normal murine fibroblast cells. Cytotoxicity was measured in vivo and survivability of mice was plotted accordingly. Therapeutic efficacy of XD was evaluated both in vivo and in vitro by determination of lipid peroxidation (LPO), reactive oxygen species (ROS) generation and by quantitating the enzyme status (GSH, catalase, superoxide dismutase) in treated and untreated samples. DNA damage was evaluated using comet and DNA fragmentation assays. Furthermore, apoptotic effect was analyzed by flow cytometry and validated by TUNEL assay and Western blotting.Results:Among all the xanthones tested XD showed IC50 at the lowest dose, and normal cells were unaffected at this dose. Survivability of mice increased significantly when treated with XD compared to other xanthones and cisplatin. Significantly increased ROS and LPO were found in cancer cells as a result of XD treatment which was unaltered in normal cell line. XD induced DNA damage and apoptosis in cancer cell lines.Interpretation & conclusions:Our experimental data indicate that XD may potentially act as a chemotherapeutic agent by enhancing ROS in breast cancer cells thereby leading to apoptosis.

Effects of Glycyrrhizin on the Differentiation of Myeloid Cells of the Heart and Lungs in Lipopolysaccharide-Induced Septic Mice.

This study investigated the effects of glycyrrhizin (GR) on the ratio of myeloid-derived suppressor cells (MDSCs) to cluster of differentiation (CD)11b+Gr1 myeloid cells in the heart and lungs in lipopolysaccharide (LPS)-induced septic mice. Mice were divided into three groups: Control, LPS, and LPS+GR. After intraperitoneal injection of phosphate-buffered saline for the Control group, LPS for the LPS group, and a combination of LPS and GR for the LPS+GR group, fluorescence-activated cell sorting was utilized to evaluate cytokines and immune cells in the blood, heart, and lungs. Histopathologic analysis of Toll-like receptor (TLR)4 was also performed. The cytokine amounts in the LPS and LPS+GR groups were significantly higher than in the Control group; however, that in the LPS+GR group was significant lower than in the LPS group. The ratio of MDSCs to CD11b+Gr1 myeloid cells in the LPS+GR group was significantly higher than in the LPS group but was significantly lower than in the Control group. The staining intensity of TLR4 showed the same pattern as that of cytokines in the heart and lungs. TLR4 staining was significantly lower in the LPS+GR group than in the LPS group but was higher than that in the Control group. GR exhibited protective effects on the heart and lungs in LPS-induced septic mice. The effects were associated with an elevated ratio of MDSCs to CD11b+Gr1 myeloid cells and the inhibition of cytokine release and TLR4 expression after GR injection.

Protective effects of systemic treatment with methylprednisolone in a rodent model of non-arteritic anterior ischemic optic neuropathy (rAION)

This study investigated the protective effects of the administration of steroids on optic nerves (ON) and retinal ganglion cells (RGCs) in a rodent model of non-arteritic anterior ischemic optic neuropathy (rAION). We induced rAION using rose bengal and argon laser irradiation in a photodynamic procedure on the optic discs of rats. The treated groups received methylprednisolone (MP) via peritoneal injection for 2 weeks. The control group received intraperitoneal injections of phosphate-buffered saline (PBS) post-rAION. At the 4th week post-infarct, MP treatments significantly rescued the RGCs (mm2) in the central retinas (1920 ± 210, p < 0.001) and mid-peripheral retinas (950 ± 240, respectively, p = 0.018) compared with those of the PBS-treated rats (central: 900 ± 210 and mid-peripheral: 440 ± 180). Functional assessment with flash visual-evoked potentials demonstrated that P1 latency (ms) was shortened in the MP group compared to the PBS group (108 ± 14 and 147 ± 9, respectively, p < 0.001). In addition, the P1 amplitude (uV) was enhanced in the MP group compared to the PBS group (55 ± 12 and 41 ± 13, respectively, p < 0.05). TUNEL assays showed a decrease in the number of apoptotic cells in the RGC layers of MP-treated retinas compared to the PBS-treated group (p < 0.05). ED1 positive cells (/HPF) were significantly decreased in the ONs of the MP group compared to the PBS group (p < 0.001). In conclusion, systemic administration of MP had neuroprotective effects on RGC survival and ON function in the rAION animal model.

Downregulation of Caveolin-1 in a Murine Model of Acute Allergic Airway Disease

Airway remodeling refers to the structural changes in the airways of asthma. Caveolin-1 reduces cell growth and negatively regulates smooth muscle cell proliferation. The aim was to investigate lung caveolin-1 status in a murine model of acute allergic airway disease. Six- to eight-week-old female BALB/c mice were sensitized by intraperitoneal injections of phosphate-buffered saline or ovalbumin (OVA) and aluminium hydroxide on Days 0 and 14, challenged with aerosolized saline or OVA (1%) on Days 21-25, 28-32, and 35. The mice were killed 1 day after the last OVA/saline challenge. Serum OVA-specific immunoglobulin E (IgE) was measured by enzyme-linked immunosorbent assay. Peribronchial inflammation was quantified by morphometric analysis. Lung caveolin-1 and Type I collagen mRNA expression was determined by real-time reverse-transcription polymerase chain reaction. Total lung collagen was measured using Sircol Assay Kit. Serum OVA-specific IgE levels were significantly elevated in OVA-challenged mice when compared with saline-challenged mice. Percentage of inflammatory cells in the bronchoalveolar lavage was significantly higher in the OVA-challenged animals. The animals' lungs that were sensitized and challenged with OVA contained large numbers of inflammatory cells concentrated near the airways and in the perivascular areas. The thickness of the bronchial epithelial layer and smooth muscle layer and the numbers of total inflammatory cells and eosinophils significantly increased in OVA-challenged mice. Caveolin-1 mRNA expression significantly decreased and Type I collagen mRNA expression significantly increased in the lung tissue of OVA-challenged mice. These results suggest that caveolin-1 seems to be involved in the pathogenesis of airway remodeling of acute allergic airway disease.

Down-regulation of Caveolin-1, an Inhibitor of Transforming Growth Factor-β Signaling, in Acute Allergen-induced Airway Remodeling

Asthma can progress to subepithelial airway fibrosis, mediated in large part by transforming growth factor-beta (TGF-beta). The scaffolding protein caveolin-1 (cav1) can inhibit the activity of TGF-beta, perhaps by forming membrane invaginations that enfold TGF-beta receptors. The study goals were 1) to evaluate how allergen challenge affects lung expression of cav1 and the density of caveolae in vivo 2) to determine whether reduced cav1 expression is mediated by interleukin (IL)-4 and 3) to measure the effects of decreased expression of cav1 on TGF-beta signaling. C57BL/6J, IL-4-deficient mice, and cav1-deficient mice, sensitized by intraperitoneal injections of phosphate-buffered saline or ovalbumin (OVA) at days 0 and 12, received intranasal phosphate-buffered saline or OVA challenges at days 24, 26, and 28. Additionally, another group of C57BL/6J mice received IL-4 by intratracheal instillation for 7 days. We confirmed that the OVA-allergen challenge increased eosinophilia and T-helper type 2-related cytokine levels (IL-4, IL-5, and IL-13) in bronchoalveolar lavage. Allergen challenge reduced lung cav1 mRNA abundance by 40%, cav1 protein by 30%, and the number of lung fibroblast caveolae by 50%. Administration of IL-4 in vivo also substantially decreased cav1 expression. In contrast, the allergen challenge did not decrease cav1 expression in IL-4-deficient mice. The reduced expression of cav1 was associated with activation of TGF-beta signaling that was further enhanced in OVA-sensitized and challenged cav1-deficient mice. This study demonstrates a previously unknown modulation of TGF-beta signaling by IL-4, via cav1, suggesting novel therapeutic targets for controlling the effects of TGF-beta and thereby ameliorating pathological airway remodeling.

An Albumin-Exendin-4 Conjugate Engages Central and Peripheral Circuits Regulating Murine Energy and Glucose Homeostasis

Glucagon-like peptide-1 (GLP-1) regulates glucose homeostasis through multiple mechanisms including direct actions on the endocrine pancreas and indirect activation of central nervous system circuits regulating gastric emptying, satiety, and body weight. Because native GLP-1 is rapidly degraded, there is considerable interest in development of more potent GLP-1 receptor (GLP-1R) agonists with sustained activity; however, the extent to which much larger GLP-1R agonists will mimic some or all of the actions of smaller peptides remains uncertain. We studied the actions of CJC-1134-PC, a recombinant human serum albumin-exendin-4 conjugated protein, at the GLP-1R using heterologous cells expressing the GLP-1R in vitro and both wild-type and Glp1r(-/-) mice in vivo. CJC-1134-PC activated GLP-1R-dependent signaling in baby hamster kidney-GLP-1R cells and acutely lowered blood glucose in wild-type but not in Glp1r(-/-) mice. Moreover, acute administration of CJC-1134-PC rapidly activated c-Fos expression in multiple regions of the central nervous system, acutely inhibited gastric emptying, and produced sustained inhibition of food intake in a GLP-1R-dependent manner. Furthermore, chronic daily treatment of high-fat diet-fed wild-type mice with CJC-1134-PC for 4 weeks led to improved glucose tolerance, increased levels of glucose-stimulated insulin, decreased HbA1c, and weight loss associated with decreased hepatic triglyceride content. These findings illustrate that a high-molecular-weight exendin-4-albumin conjugate retains the ability to mimic a full spectrum of GLP-1R-dependent actions, including activation of central nervous system circuits regulating gastric emptying, food intake, and body weight.

Inhibition of In Vivo Tumor Angiogenesis and Growth Via Systemic Delivery of an Angiopoietin 2-Specific RNA Aptamer

Cellular events mediated by the Tie2 receptor are important to tumor neovascularization. Despite the complex interplay of the best-characterized Tie2 ligands, angiopoietins 1 and 2, Ang2 is purportedly "proangiogenic" in the presence of vascular endothelial growth factor. We examined whether in vivo administration of an RNA aptamer that specifically blocks Ang 2 would inhibit tumor angiogenesis and growth. Ang2-mediated Tie2 receptor phosphorylation was assessed in vitro in the absence and presence of aptamer coupled to polyethylene glycol. IN VIVO ANGIOGENESIS ASSAY: CT26 murine colon carcinoma cells expressing green fluorescent protein were delivered into mouse dorsal skinfold window chambers. Animals received daily intraperitoneal injections of phosphate-buffered saline, low-dose (Ang2 aptamer-LD; 1 mg/kg/d), or high-dose aptamer (Ang2 aptamer-HD; 10 mg/kg/d). Vascular length density was measured under fluorescence microscopy. PRIMARY TUMOR GROWTH: CT26 cells expressing luciferase were injected into flanks of BALB/c mice to allow tumor growth monitoring by bioluminescence imaging. Animals received continuous phosphate-buffered saline or aptamer (1 mg/kg/d) via ALZET pumps. Tumors were assessed for CD31/PECAM-1 immunostaining and Hoechst dye uptake. Pegylated aptamer inhibited Tie2 phosphorylation. Systemic aptamer administration reduced vascular length density (P < or = 0.03) and decreased bioluminescence emission (P < 0.04), corresponding to 50% decrease in tumor volume (P = 0.04). Control tumors displayed abundant vascular marker staining, in contrast to tumors from aptamer-treated animals. in vivo administration of a clinically relevant, pegylated RNA aptamer specifically designed against Ang2 inhibited tumor angiogenesis and growth. These findings support targeted Ang2 inhibition as a relevant anti-angiogenic, anti-neoplastic strategy.

Inhibition of Choroidal Neovascularization by a Peptide Inhibitor ofthe Urokinase Plasminogen Activator and Receptor System in a Mouse Model

To determine the role played by the urokinase plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR) system in choroidal neovascularization (CNV) and whether inhibition of this system can suppress the extent of CNV in an animal model. Choroidal neovascularization was induced in mice by laser photocoagulation using the slitlamp delivery system. Reverse transcriptase-polymerase chain reaction and immunocytochemical analysis were performed on the retina choroids of these animals to examine the expression of uPAR. For 2 weeks following laser treatment, animals were injected intraperitoneally with a novel peptide inhibitor of the uPA-uPAR system (100 mg/kg twice a day every day, every other day, and once a week). Control laser-treated animals receive an intraperitoneal injection of phosphate-buffered saline every day. Following treatment, animals were perfused with fluorescein-labeled dextran, eyes were removed, and the areas of new vessels were examined in the retina-choroid whole mounts by fluorescence microscopy and quantitated using image analysis software. In this study, uPAR was found to be up-regulated in the choroidal tissues of mice with laser-induced CNV. The uPAR was localized to the endothelial cells of the fibrovascular tissue within the CNV complex. Systemic administration of the peptide inhibitor of the uPA-uPAR system resulted in a significant reduction of CNV (up to 94%). The response was found to be frequency-of-dose dependent. No toxic effects or tissue destruction was noted following the peptide treatment. Our results strongly suggest that up-regulation of the uPA-uPAR system is an important step during CNV, and significant inhibition of CNV was seen when cell surface-associated uPA-uPAR activity was prevented with the peptide inhibitor. Clinical Relevance Inhibition of the protease system (uPA-uPAR) may prove to be a potential novel antiangiogenic therapy for CNV as seen in age-related macular degeneration.

Epidermal growth factor accelerates connective tissue wound healing in the perforated rat mesentery.

Epidermal growth factor (EGF) has been reported to stimulate healing of wounds in skin, cornea, and gastric mucosa. In the present study, we further investigate the effect of endogenous and exogenous EGF in healing of connective tissue wounds using the rat perforated mesentery model. Healing of mesenteric perforations is accomplished by the connective tissue fibroblasts since there are no interfering variables such as interactions of epithelial cells, desiccation, or foreign materials such as sutures or subcutaneous implants. We performed laparotomy in 114 adult male Sprague-Dawley rats and made 20 standardized perforations in the mesentery of each rat with a scalpel. Rats were randomly assigned to five groups. Group I received no treatment after surgery; Group II received intraperitoneal injections of phosphate-buffered saline (PBS) after surgery and then twice daily for the following 3 days; Group III received 10 micrograms of EGF in the PBS injections according to the same regimen as Group II; Group IV had sham exploration of the submandibular salivary glands; and Group V animals had excision of the submandibular glands 3 days before laparotomy to deprive the main source of EGF in rat. On Days 4 through 10 after surgery rats were sacrificed and the percentage of perforations in each rat which were closed was determined. The curves for the time course of wound closure for Groups IV and V were not different indicating that endogenous submandibular EGF does not play a role in healing of mesenteric wounds.(ABSTRACT TRUNCATED AT 250 WORDS)